Highly-multiplexed imaging by Cyclic Immunofluorescence (CycIF) of protein levels and protein phosphorylation states in the MCF 10A breast cell line treated with EGF or four kinase inhibitors
- Dataset
(ID:20267)

Highly-multiplexed imaging by Cyclic Immunofluorescence (CycIF) of protein levels and protein phosphorylation states in the MCF 10A breast cell line treated with EGF or four kinase inhibitors

Screening Lab Investigator:

Jia-Ren Lin

Screening Principal Investigator:

Peter K. Sorger

Assay Description:

Cyclic Immunofluorescence (CycIF) is a fluorescence imaging assay used for multiplexed measurement of protein levels and protein phosphorylation states in single cells. The MCF 10A breast cell line was plated in 96-well plates and treated with different doses of one of five perturbagens (EGF and the kinase inhibitors Lapatinib, Selumetinib, Dactolisib, and Torkinib) for 24 hr before being fixed and subjected to nine cycles of CycIF staining according to the protocol described below.

Assay Protocol:

1. MCF 10A cells were plated at 5,000 cells per well in duplicate 96-well plates and grown for 48 hr to 70-80% confluency. Cell growth conditions are reported in PMID: 27135972. Cells that were to be treated with EGF were grown under the reported conditions for 24 hr and then serum starved for 24 hr.
2. Following this initial growth period, the cells were treated with the indicated doses of EGF, Lapatinib, Selumetinib, Dactolisib, or Torkinib for 24 hrs.
3. After treatment, cells in each well were fixed with 140 uL 4% PFA at room temperature for 30 min and then washed 4 times with 250 uL PBS per wash using a Bio-Tek EL406 plate washer.
4. To permeabilize the cells, 140 uL ice-cold methanol were added, and the plates were left at room temperature for 10 minutes. The cells then were washed 4 times with 250 uL PBS per wash as above.
5. The permeabilized cells were blocked by incubation with 50 uL Odyssey blocking buffer at room temperature for 1 hr.
6. Primary antibodies were diluted in 50 uL Odyssey buffer and added to the cells. The cells were incubated overnight at 4°C and then washed gently 4 times with 250 uL PBS per wash using the same Bio-Tek EL406 plate washer on the lowest setting. If the primary antibody was unlabeled, diluted secondary antibody then was added to the cells, and the cells were incubated for 1 hr at room temperature and washed as just described.
7. Hoechst 33342 (1 mg/mL stock) was diluted 1:5000 in 140 uL PBS and incubated with the cells for 15 minutes at room temperature, and the cells again were washed gently 4 times with 250 uL PBS.
8. To image the fluorescent signals, a Cytell imager (GE Healthcare Life Sciences) was used with a 10x objective lens and fixed filter setting for 4-channel immunofluorescence (Hoechst, 488 nm, 555 nm, and 647 nm) to minimize crosstalk. Weak signals from fluorophore-conjugated primary antibodies required a binning option or high-sensitivity camera (EMCCD, sCMOS).
9. To inactivate the fluorophore(s) used in the first round of imaging, the PBS in each well was removed, 140 uL of fluorophore-inactivation solution were added, and the plates were incubated at room temperature for 1 hr with light exposure from a tabletop lamp or similar. Each well then was washed gently 4 times with 250 uL PBS using a plate washer as described above.
10. After fluorophore inactivation, the cells were imaged again to ensure complete removal of fluorescent signal.
11. The cells then were subjected to eight more rounds of labeling by repeating the blocking (step 5) through fluorophore inactivation (step 9) steps described above.
12. To align the images from all rounds of labeling, the Hoechst images from each round were used as reference images and registered using a rigid body transformation function. The same registration information then was used to align the images from the other fluorescent channels (488 nm, 555 nm, and 647 nm).
13. Image segmentation and analysis were performed using ImageJ and the scripts available at lincs.hms.harvard.edu/lin-natcommun-2015/. In brief, the Hoechst images were converted to nuclear masks. Nuclear masks were converted into RING regions of interest (ROIs) and used to define the quantification areas for the images in each of the other fluorescent channels. For fluorescent intensity measurement, the raw images were first background subtracted using the rolling ball method with a 50-pixel radius. Then, the nuclear masks/ROIs were applied to each image and the intensity (reported in arbitrary units) measured. Cell area, perimeter, and circularity also were calculated using default functions in ImageJ.
14. Between 500 and 3500 cells per condition were analyzed, and the x-y coordinates for each analyzed cell in the registered images are reported in the accompanying dataset. The complete set of images is viewable online through our OMERO server lincs-omero.hms.harvard.edu/webclient/?show=dataset-1602. Lookup tables for antibody/CycIF information and for data file data column definitions also are contained within the downloadable data package for this dataset.